M3 Muscarinic Acetylcholine Receptor Antagonists

ABSTRACT

Muscarinic Acetylcholine receptor antagonists and methods of using them are provided.

This application is a continuation application of U.S. Ser. No.11/774,885, filed 9 Jul. 2007, now allowed, which is a continuation ofU.S. Ser. No. 10/577,834, now U.S. Pat. No. 7,439,255, filed 1 May 2006,which is the §371 national stage entry of PCT/US2004/036663, filed 4Nov. 2004 and which claims the benefit of priority from U.S. Ser. No.60/517243, filed 4 Nov. 2003.

FIELD OF THE INVENTION

This invention relates to novel derivatives of 8-azoniabicyclo[3,2,1]octanes, pharmaceutical compositions, processes for theirpreparation, and use thereof in treating M₃ muscarinic acetylcholinereceptor mediated diseases.

BACKGROUND OF THE INVENTION

Acetylcholine released from cholinergic neurons in the peripheral andcentral nervous systems affects many different biological processesthrough interaction with two major classes of acetylcholinereceptors—the nicotinic and the muscarinic acetylcholine receptors.Muscarinic acetylcholine receptors (mAChRs) belong to the superfamily ofG-protein coupled receptors that have seven transmembrane domains. Thereare five subtypes of mAChRs, termed M₁-M₅, and each is the product of adistinct gene. Each of these five subtypes displays uniquepharmacological properties. Muscarinic acetylcholine receptors arewidely distributed in vertebrate organs where they mediate many of thevital functions. Muscarinic receptors can mediate both inhibitory andexcitatory actions. For example, in smooth muscle found in the airways,M₃ mAChRs mediate contractile responses. For review, please seeCaulfield (1993 Pharmac. Ther. 58:319-79).

In the lungs, mAChRs have been localized to smooth muscle in the tracheaand bronchi, the submucosal glands, and the parasympathetic ganglia.Muscarinic receptor density is greatest in parasympathetic ganglia andthen decreases in density from the submucosal glands to tracheal andthen bronchial smooth muscle. Muscarinic receptors are nearly absentfrom the alveoli. For review of mAChR expression and function in thelungs, please see Fryer and Jacoby (1998 Am J Respir Crit Care Med158(5, pt 3) S 154-60).

Three subtypes of mAChRs have been identified as important in the lungs,M₁, M₂ and M₃ mAChRs. The M₃ mAChRs, located on airway smooth muscle,mediate muscle contraction. Stimulation of M₃ mAChRs activates theenzyme phospholipase C via binding of the stimulatory G protein Gq/11(Gs), leading to liberation of phosphatidyl inositol-4,5-bisphosphate,resulting in phosphorylation of contractile proteins. M₃ mAChRs are alsofound on pulmonary submucosal glands. Stimulation of this population ofM₃ mAChRs results in mucus secretion.

M₂ mAChRs make up approximately 50-80% of the cholinergic receptorpopulation on airway smooth muscles. Although the precise function isstill unknown, they inhibit catecholaminergic relaxation of airwaysmooth muscle via inhibition of cAMP generation. Neuronal M₂ mAChRs arelocated on postganglionic parasympathetic nerves. Under normalphysiologic conditions, neuronal M₂ mAChRs provide tight control ofacetylcholine release from parasympathetic nerves. Inhibitory M₂ mAChRshave also been demonstrated on sympathetic nerves in the lungs of somespecies. These receptors inhibit release of noradrenaline, thusdecreasing sympathetic input to the lungs.

M₁ mAChRs are found in the pulmonary parasympathetic ganglia where theyfunction to enhance neurotransmission. These receptors have also beenlocalized to the peripheral lung parenchyma, however their function inthe parenchyma is unknown.

Muscarinic acetylcholine receptor dysfunction in the lungs has beennoted in a variety of different pathophysiological states. Inparticular, in asthma and chronic obstructive pulmonary disease (COPD),inflammatory conditions lead to loss of inhibitory M₂ muscarinicacetylcholine autoreceptor function on parasympathetic nerves supplyingthe pulmonary smooth muscle, causing increased acetylcholine releasefollowing vagal nerve stimulation (Fryer et al. 1999 Life Sci 64 (6-7)449-55). This mAChR dysfunction results in airway hyperreactivity andhyperresponsiveness mediated by increased stimulation of M₃ mAChRs. Thusthe identification of potent mAChR antagonists would be useful astherapeutics in these mAChR-mediated disease states.

COPD is an imprecise term that encompasses a variety of progressivehealth problems including chronic bronchitis, chronic bronchiolitis andemphysema, and it is a major cause of mortality and morbidity in theworld. Smoking is the major risk factor for the development of COPD;nearly 50 million people in the U.S. alone smoke cigarettes, and anestimated 3,000 people take up the habit daily. As a result, COPD isexpected to rank among the top five as a world-wide health burden by theyear 2020. Inhaled anti-cholinergic therapy is currently considered the“gold standard” as first line therapy for COPD (Pauwels et al. 2001 Am.J. Respir. Crit. Care Med. 163:1256-1276).

Despite the large body of evidence supporting the use ofanti-cholinergic therapy for the treatment of airway hyperreactivediseases, relatively few anti-cholinergic compounds are available foruse in the clinic for pulmonary indications. More specifically, inUnited States, Ipratropium Bromide (Atrovent©; and Combivent©, incombination with albuterol) is currently the only inhaledanti-cholinergic marketed for the treatment of airway hyperreactivediseases. While this compound is a potent anti-muscarinic agent, it isshort acting, and thus must be administered as many as four times dailyin order to provide relief for the COPD patient. In Europe and Asia, thelong-acting anti-cholinergic Tiotropium Bromide (Spiriva©) was recentlyapproved, however this product is currently not available in the UnitedStates. Thus, there remains a need for novel compounds that are capableof causing blockade at mAChRs which are long acting and can beadministered once-daily for the treatment of airway hyperreactivediseases such as asthma and COPD.

Since mAChRs are widely distributed throughout the body, the ability toapply anti-cholinergics locally and/or topically to the respiratorytract is particularly advantageous, as it would allow for lower doses ofthe drug to be utilized. Furthermore, the ability to design topicallyactive drugs that have long duration of action, and in particular, areretained either at the receptor or by the lung, would allow theavoidance of unwanted side effects that may be seen with systemicanti-cholinergic use.

SUMMARY OF THE INVENTION

This invention provides for a method of treating a muscarinicacetylcholine receptor (mAChR) mediated disease, wherein acetylcholinebinds to an M₃ mAChR and which method comprises administering aneffective amount of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof.

This invention also relates to a method of inhibiting the binding ofacetylcholine to its receptors in a mammal in need thereof whichcomprises administering to aforementioned mammal an effective amount ofa compound of Formula (I).

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to novel derivatives of 8-azoniabicyclo[3,2,1]octanes, pharmaceutical compositions, processes for theirpreparation, and use thereof in treating M₃ muscarinic acetylcholinereceptor mediated diseases, represented by Formula (I)

wherein:

-   the H atom indicated is in the exo position;-   R1⁻ represents an anion associated with the positive charge of the N    atom. R1⁻ may be but is not limited to chloride, bromide, iodide,    sulfate, benzene sulfonate and toluene sulfonate;-   R2 is selected from the group consisting of straight or branched    chain lower alkyl groups (having preferably from 1 to 6 carbon    atoms), cycloalkyl groups (having from 5 to 6 carbon atoms),    cycloalkyl-alkyl (having 6 to 10 carbon atoms), heterocycloalkyl    (having 5 to 6 carbon atoms) and N or O as the heteroatom,    heterocycloalkyl-alkyl (having 6 to 10 carbon atoms) and N or O as    the heteroatom, aryl, optionally substituted aryl, heteroaryl, and    optionally substituted heteroaryl;-   R3 is selected from the group consisting of (C₂-C₁₂)alkyl,    (C₁-C₆)alkenyl, (C₁-C₆)alkyl(C₃-C₆)cycloalkyl, (C₁-C₆)alkyl-phenyl,    (C₁-C₆)alkyl-OH, (C₁-C₆)alkyl-CN, (C₁-C₆)alkyl-halogen,    (C₁-C₆)alkyl-CF₃, (C₁-C₆)alkyl-OCH₃,    (C₁-C₆)alkyl-O-(C₁-C₆)alkyl-OCH₃ and (C₁-C₆)alkyl-O-(C₁-C₆)aryl.    Preferred R3 substituents are in the endo position.

All of the aryl, heteroaryl, and heterocyclic containing moieties may beoptionally substituted as defined herein below.

For use herein the term “the aryl, heteroaryl, and heterocycliccontaining moieties” refers to both the ring and the alkyl, or ifincluded, the alkenyl rings, such as aryl, arylalkyl, and aryl alkenylrings. The term “moieties” and “rings” may be interchangeably usedthroughout.

As used herein, “optionally substituted” unless specifically definedshall mean such groups as halogen, such as fluorine, chlorine, bromineor iodine; hydroxy; hydroxy substituted C₁₋₁₀alkyl; C₁₋₁₀ alkoxy, suchas methoxy or ethoxy; S(O)_(m′)C₁₋₁₀ alkyl, wherein m′ is 0, 1 or 2,such as methyl thio, methyl sulfinyl or methyl sulfonyl; amino, mono &di-substituted amino, such as in the NR₄R₅ group; NHC(O)R₄; C(O)NR₄R₅;C(O)OH; S(O)₂NR₄R₅; NHS(O)₂R₄, C₁₋₁₀ alkyl, such as methyl, ethyl,propyl, isopropyl, or t-butyl; halosubstituted C₁₋₁₀ alkyl, such as CF₃;an optionally substituted aryl, such as phenyl, or an optionallysubstituted arylalkyl, such as benzyl or phenethyl, optionallysubstituted heterocylic, optionally substituted heterocyclicalkyl,optionally substituted heteroaryl, optionally substituted heteroarylalkyl, wherein these aryl, heteroaryl, or heterocyclic moieties may besubstituted one to two times by halogen; hydroxy; hydroxy substitutedalkyl; C₁₋₁₀ alkoxy; S(O)_(m′)C₁₋₁₀ alkyl; amino, mono & di-substitutedalkyl amino, such as in the NR₄R₅ group; C₁₋₁₀ alkyl, or halosubstitutedC₁₋₁₀ alkyl, such as CF₃.

Suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of inorganic and organicacids, such as hydrochloric acid, hydrobromic acid, sulphuric acid,phosphoric acid, methane sulphonic acid, ethane sulphonic acid, aceticacid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid,succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid,phenylacetic acid and mandelic acid. In addition, pharmaceuticallyacceptable salts of compounds of Formula (I) may also be formed with apharmaceutically acceptable cation. Suitable pharmaceutically acceptablecations are well known to those skilled in the art and include alkaline,alkaline earth, ammonium and quaternary ammonium cations.

The following terms, as used herein, refer to:

-   -   “halo”—all halogens, that is chloro, fluoro, bromo and iodo.    -   “C₁₋₁₀alkyl” or “alkyl”—both straight and branched chain        moieties of 1 to 10 carbon atoms, unless the chain length is        otherwise limited, including, but not limited to, methyl, ethyl,        n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl,        n-pentyl and the like.    -   “cycloalkyl” is used herein to mean cyclic moiety, preferably of        3 to 8 carbons, including but not limited to cyclopropyl,        cyclopentyl, cyclohexyl, and the like.    -   “alkenyl” is used herein at all occurrences to mean straight or        branched chain moiety of 2-10 carbon atoms, unless the chain        length is limited thereto, including, but not limited to        ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl,        2-butenyl and the like.    -   “aryl”—phenyl and naphthyl;    -   “heteroaryl” (on its own or in any combination, such as        “heteroaryloxy”, or “heteroaryl alkyl”)—a 5-10 membered aromatic        ring system in which one or more rings contain one or more        heteroatoms selected from the group consisting of N, O or S,        such as, but not limited, to pyrrole, pyrazole, furan,        thiophene, quinoline, isoquinoline, quinazolinyl, pyridine,        pyrimidine, oxazole, tetrazole, thiazole, thiadiazole, triazole,        imidazole, or benzimidazole.    -   “heterocyclic” (on its own or in any combination, such as        “heterocyclicalkyl” or “heterocycloalkyl”)—a saturated or        partially unsaturated 4-10 membered ring system in which one or        more rings contain one or more heteroatoms selected from the        group consisting of N, O, or S; such as, but not limited to,        pyrrolidine, piperidine, piperazine, morpholine,        tetrahydropyran, thiomorpholine, or imidazolidine. Furthermore,        sulfur may be optionally oxidized to the sulfone or the        sulfoxide.    -   “arylalkyl” or “heteroarylalkyl” or “heterocyclicalkyl” is used        herein to mean C₁₋₁₀ alkyl, as defined above, attached to an        aryl, heteroaryl or heterocyclic moiety, as also defined herein,        unless otherwise indicated.    -   “sulfinyl”—the oxide S (O) of the corresponding sulfide, the        term “thio” refers to the sulfide, and the term “sulfonyl”        refers to the fully oxidized S(O)₂ moiety.    -   Preferred compounds useful in the present invention include:

-   (3-Endo)-3-(2-cyano-2,2-diphenylethyl)-8-(cyclohexylmethyl)-8-methyl-8-azoniabicyclo[3.2.1]octane    bromide;

-   (3-Endo)-3-(2-cyano-2,2-diphenylethyl)-8-(cyclopropylmethyl)-8-methyl-8-azoniabicyclo[3.2.1]octane    bromide;

-   (3-Endo)-8-butyl-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-azoniabicyclo[3.2.1]octane    bromide;

-   (3-Endo)-8-(4-chlorobutyl)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-azoniabicyclo[3.2.1]octane    bromide;

-   (3-Endo)-3-(2-cyano-2,2-diphenylethyl)-8-dodecyl-8-methyl-8-azoniabicyclo[3.2.1]octane    bromide;

-   (3-Endo)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-(2-propen-1-yl)-8-azoniabicyclo[3.2.1]octane    iodide;

-   (3-Endo)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-(phenylmethyl)-8-azoniabicyclo[3.2.1]octane    bromide;

-   (3-Endo)-3-(2-cyano-2,2-diphenylethyl)-8-(2-hydroxyethyl)-8-methyl-8-azoniabicyclo[3.2.1]octane    bromide;

-   (3-Endo)-3-(2-cyano-2,2-diphenylethyl)-8-ethyl-8-methyl-8-azoniabicyclo[3.2.1]octane    bromide;

-   (3-Endo)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-propyl-8-azoniabicyclo[3.2.1]octane    bromide;

-   (3-Endo)-3-(2-cyano-2,2-di    phenylethyl)-8-(5-hexen-1-yl)-8-methyl-8-azoniabicyclo[3.2.1]octane    bromide;

-   (3-Endo)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-(4,4,4-trifluorobutyl)-8-azoniabicyclo[3.2.1]octane    bromide;

-   (3-Endo)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-(3-phenylpropyl)-8-azoniabicyclo[3.2.1]octane    bromide;

-   (3-Endo)-3-(2-cyano-2,2-diphenylethyl)-8-(2-cyclohexylethyl)-8-methyl-8-azoniabicyclo[3.2.1]octane    bromide;

-   (3-Endo)-3-(2-cyano-2,2-diphenylethyl)-8-(3-cyanopropyl)-8-methyl-8-azoniabicyclo[3.2.1]octane    bromide;

-   (3-Endo)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-[2-(methyloxy)ethyl]-8-azoniabicyclo[3.2.1]octane    bromide;

-   (3-Endo)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-(2-{[2-(methyloxy)ethyl]oxy}ethyl)-8-azoniabicyclo[3.2.1]octane    bromide;

-   N-(Endo)-(3-endo)-3-(2-cyano-2,2-diphenylethyl)-(8-endo)-8-(5-hexen-1-yl)-8-methyl-8-azoniabicyclo[3.2.1]octane    bromide;

-   N-(Endo)-(3-endo)-(2-cyano-2,2-diphenylethyl)-(8-endo)-methyl-8-{2-[(phenylmethyl)oxy]ethyl}-8-azoniabicyclo[3.2.1]octane    bromide;

-   N-(Endo)-(3-endo)-(2-cyano-2,2-diphenylethyl)-8-methyl-8-(3-phenylpropyl)-8-azoniabicyclo[3.2.1]    octane bromide; and

-   N-(Endo)-(3-endo)-(2-cyano-2,2-diphenylethyl)-8-methyl-8-[3-(phenyloxy)propyl]-8-azoniabicyclo[3.2.1]octane    bromide.

Methods of Preparation

Preparation

The compounds of Formula (I) may be obtained by applying syntheticprocedures, some of which are illustrated in the Schemes below. Thesynthesis provided for these Schemes is applicable for producingcompounds of Formula (I) having a variety of different R1, R2 and R3which are reacted, employing substituents which are suitable protected,to achieve compatibility with the reactions outlined herein. Subsequentdeprotection, in those cases, then affords compounds of the naturegenerally disclosed. While some Schemes are shown with specificcompounds, this is merely for illustration purpose only.

A general preparation method is shown in Scheme I. The synthesis startedwith compound 1. Coupling reaction with the anion derived fromHC(CN)(R2)(R2) provided 2. Treatment with R3-R1 then furnished thequaternary ammonium salt with Formula (I).

A more specific preparation method leading to compounds with Formula (I)is outlined in Scheme II. Alkylation of diphenylacetonitrile with 1afforded compound 3. Treatment with CF₃(CH₂)₃Br then affordedquarternary ammonium salt 4.

A specific route to N-endo compound of Formula (I) is exemplified inScheme III. Treatment of alcohol 5 with iodine and plymer supportetriphenyl phosphine gives the iodide 6 which in turn can be converted tocompound 7 by reaction with a disbustituted acetonitrile derivative 13under basic conditions. Removal of the benzyl group of 7 leads to thesecondary amine 8. Sequential N-alkylation of 8 with R3-R1 and methylbromide affords the quaternary ammonium salt 10 of Formula (I) asmixture of diastereosisomers. Recrystallisation in an organic solventmixture comprised of solvent such DCM, EA or hexane affords the pureN-endo isomer 11 of formula (I).

Alternatively the secondary amine 8 can de prepared from the N-Bocprotected alcohol derivative 14 by generation of a tosylate derivativewhich can be displaced by the disubstituted acetonirile 13 withconcomitant removal of the BOC group as depicted in Scheme IV.

SYNTHETIC EXAMPLES

The invention will now be described by reference to the followingExamples which are merely illustrative and are not to be construed as alimitation of the scope of the present invention. Most reagents andintermediates are commercially available or are prepared according toprocedures in the literature. The preparation of intermediates notdescribed in the literature is also illustrated below.

Flash column chromatography was carried out using Merck 9385 silicaunless stated otherwise.

LC/MS analyses were conducted under the following conditions:

-   -   Column: 3.3cm×4.6 mm ID, 3 um ABZ+PLUS    -   Flow Rate: 3 ml/min    -   Injection Volume: 5 μl    -   Temp: Room temperature    -   Solvents:        -   A: 0.1% Formic Acid+10 m Molar Ammonium Acetate.        -   B: 95% Acetonitrile+0.05% Formic Acid

Gradient: Time A % B % 0.00 100 0 0.70 100 0 4.20 0 100 5.30 0 100 5.50100 0

The Gilson preparatory HPLC was conducted under the followingconditions:

-   -   Column: 75×33 mm I.D., S-5 um, 12 nm    -   Flow rate: 30 mL/min    -   Injection Volume: 0.800 mL    -   Room temperature    -   Solvent A: 0.1% trifluoroacetic acid in water    -   Solvent B: 0.1% trifluoroacetic acid in acetonitrile

Intermediate 1: Preparation of3-((endo)-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-2,2-diphenyl-propionitrile

a) Preparation of((endo)-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-methanol

A mixture of 1,1-dimethylethyl(endo)-3-(hydroxymethyl)-8-azabicyclo[3.2.1] octane-8-carboxylate (0.50g, 2.05 mmol) and LiAlH₄ (6.16 mL, 1.0 M in THF, 6.16 mmol) was heatedat 80° C. in a microwave reactor for 60 min. The solution was then mixedwith saturated Na₂SO₄ solution, filtered through celite and concentratedto afford the title compound (0.31 g, 97%): LCMS (ES) m/z 156 (M+H)⁺;¹H-NMR(CDCl₃) δ 1.28 (s, 1H), 1.59 (m, 4H), 1.90 (m, 1H), 2.13 (m, 4H),2.32 (s, 3H), 3.17 (s, 2H), 3.59 (d, 2H).

b) Preparation of(endo)-3-iodomethyl-8-methyl-8-aza-bicyclo[3.2.1]octane

A solution of iodine (6.67 g, 25.8 mmol) and((endo)-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-methanol (2.0 g, 12.9mmol) in CH₂Cl₂ (120 mL) was mixed with PPh₃ (on resin, 8.6 g, 3 mmol/g,25.8 mmol). The resultant mixture was stirred for 17 hours, filtered andconcentrated to afford the title compound (2.63 g, 77%): LCMS (ES) m/z266 (M+H)⁺; ¹H-NMR(CDCl₃) δ 2.05 (m, 4H), 2.39 (m, 3H), 2.79 (d, 3H),2.98 (m, 2H), 3.45 (d, 2H), 3.81 (s, 2H).

c) Preparation of3-((endo)-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-2,2-diphenyl-propionitrile

A solution of(endo)-3-iodomethyl-8-methyl-8-aza-bicyclo[3.2.1]octane(1.06 g, 4.0mmol) and Ph₂CHCN (2.32 g, 12.0 mmol) in DMF (20 mL) was mixed with NaH(0.288 g, 12.0 mmol). The resultant mixture was stirred at roomtemperature for 60 minutes. Filtration and purification via a reversephase HPLC (Gilson) then afforded the title compound (1.16 g, 93%): LCMS(ES) m/z 331 (M+H)⁺; ¹H-NMR(CDCl₃) δ 1.64 (m, 2H), 2.14 (m, 1H), 2.26(m, 2H), 2.34 (m, 2H), 2.52 (m, 2H), 2.75 (m, 5H), 3.83 (s, 2H), 7.39(d, 10H).

General Preparation Procedures

A solution of3-((3-endo)-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-2,2-diphenyl-propionitrile(33.0 mg, 0.10 mmol) in CH₂Cl₂ (0.5 mL) and MeCN (0.5 mL) was mixed withRBr (1.0 mmol) and K₂CO₃ (27.6 mg, 0.20 mmol). The resultant mixture wasstirred at room temperature for certain reaction time (specified infollowing examples). It was then diluted with DMSO (0.3 mL) andconcentrated. Purification via a reverse phase HPLC (Gilson) affordedthe target compound.

Example 1

(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8-(cyclohexylmethyl)-8-methyl-8-azoniabicyclo[3.2.1]octanebromide.

The title compound was prepared in 15% yield by following the generalexperimental procedure (reaction time=7 days): LCMS (ES) m/z 427 (M)⁺;¹H-NMR(CDCl₃) δ 1.29 (m, 3H), 1.43 (m, 2H), 1.83 (m, 8H), 2.19 (m, 1H),2.42 (m, 6H), 3.00 (m, 2H), 3.04 (s, 3H), 3.10 (d, 2H), 3.84 (s, 2H),7.35 (m, 2H), 7.43 (m, 4H), 7.49 (m, 4H).

Example 2

(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8-(cyclopropylmethyl)-8-methyl-8-azoniabicyclo[3.2.1]octanebromide

The title compound was prepared in 51% yield by following the generalexperimental procedure (reaction time=70 hours): LCMS (ES) m/z 385 (M)⁺;¹H-NMR(CDCl₃) δ 0.48 (m, 2H), 0.83 (m, 2H), 1.13 (m, 1H), 1.82 (m, 2H),2.22 (m, 1H), 2.42 (m, 6H), 3.01 (m, 2H), 3.12 (m, 3H), 3.19 (d, 2H),3.90 (m, 2H), 7.35 (m, 2H), 7.43 (m, 4H), 7.50 (m, 4H).

Example 3

(3-endo)-8-butyl-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-azoniabicyclo[3.2.1]octanebromide

The title compound was prepared in 26% yield by following the generalexperimental procedure (reaction time=70 hours): LCMS (ES) m/z 387 (M)⁺;¹H-NMR(CDCl₃) δ 1.04 (m, 3H), 1.45 (m, 2H), 1.74 (m, 2H), 1.84 (d, 2H),2.21 (m, 1H), 2.45 (m, 6H), 3.00 (m, 2H), 3.02 (s, 3H), 3.20 (m, 2H),3.83 (s, 2H), 7.35 (m, 2H), 7.42 (m, 4H), 7.49 (m, 4H).

Example 4

(3-endo)-8-(4-chlorobutyl)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-azoniabicyclo[3.2.1]octanebromide

The title compound was prepared in 37% yield by following the generalexperimental procedure (reaction time=70 hours): LCMS (ES) m/z 421 (M)⁺;¹H-NMR(CDCl₃) δ 1.88 (m, 6H), 2.18 (m, 1H), 2.45 (m, 6H), 3.01 (m, 2H),3.04 (s, 3H), 3.28 (m, 2H), 3.67 (m, 2H), 3.84 (s, 2H), 7.35 (m, 2H),7.42 (m, 4H), 7.49 (m, 4H).

Example 5

(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8-dodecyl-8-methyl-8-azoniabicyclo[3.2.1]octanebromide

The title compound was prepared in 10% yield by following the generalexperimental procedure (reaction time=70 hours): LCMS (ES) m/z 499 (M)⁺;¹H-NMR(CDCl₃) δ 0.95 (t, 3H), 1.36 (m, 18H), 1.73 (m, 2H), 1.82 (d, 2H),2.18 (m, 1H), 2.46 (m, 6H), 3.00 (d, 2H), 3.02 (s, 3H), 3.19 (m, 2H),3.82 (s, 2H), 7.35 (m, 2H), 7.42 (m, 4H), 7.49 (m, 4H).

Example 6

(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-(2-propen-1-yl)-8-azoniabicyclo[3.2.1]octaneiodide

The title compound was prepared in 37% yield by following the generalexperimental procedure (reaction time=3 hours): LCMS (ES) m/z 371 (M)⁺;¹H-NMR(CDCl₃) δ 1.83 (m, 2H), 2.20 (m, 1 H), 2.36 (m, 2H), 2.47 (m, 4H),3.02 (m, 5H), 3.85 (s, 2H), 3.92 (d, 2H), 5.71 (m, 2H), 6.09 (m, 1 H),7.35 (m, 2H), 7.42 (m, 4H), 7.51 (m, 4H).

Example 7

(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-(phenylmethyl)-8-azoniabicyclo[3.2.1]octanebromide

The title compound was prepared in 39% yield by following the generalexperimental procedure (reaction time=3 hours): LCMS (ES) m/z 421 (M)⁺;¹H-NMR(CDCl₃) δ 1.85 (d, 2H), 2.17 (m, 1 H), 2.45 (m, 4H), 2.73 (m, 2H),2.92 (s, 3H), 3.04 (d, 2H), 3.86 (s, 2H), 4.45 (s, 2H), 7.34 (m, 2H),7.42 (m, 4H), 7.48 (m, 4H), 7.56 (m, 5H).

Example 8

(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8-(2-hydroxyethyl)-8-methyl-8-azoniabicyclo[3.2.1]octanebromide

The title compound was prepared in 40% yield by following the generalexperimental procedure (reaction time=10 days): LCMS (ES) m/z 375 (M)⁺;¹H-NMR(CDCl₃) δ 1.84 (m, 2H), 2.04 (m, 1 H), 2.22 (m, 2H), 2.34 (m, 2H),2.50 (m, 2H), 2.74 (s, 1 H), 2.95 (d, 1H), 3.01 (d, 1H), 3.14 (s,1 H),3.33 (s, 3H), 3.40 (m, 1H), 3.81 (m, 1H), 3.99 (m, 2H), 7.35 (m, 2H),7.42 (m, 4H), 7.48 (m, 4H).

Example 9

(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8-ethyl-8-methyl-8-azoniabicyclo[3.2.1]octanebromide

The title compound was prepared in 60% yield by following the generalexperimental procedure (reaction time=70 hours): LCMS (ES) m/z 359 (M)⁺;¹H-NMR(CDCl₃) δ 1.32 (t, 3H), 1.83 (d, 2H), 2.21 (m, 1H), 2.36 (m, 4H),2.52 (m, 2H), 3.02 (m, 5H), 3.35 (m, 2H), 3.81 (s, 2H), 7.35 (m, 2H),7.42 (m, 4H), 7.49 (m, 4H).

Example 10

(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-propyl-8-azoniabicyclo[3.2.1]octanebromide

The title compound was prepared in 20% yield by following the generalexperimental procedure (reaction time=70 hours): LCMS (ES) m/z 373 (M)⁺;¹H-NMR(CDCl₃) δ 1.02 (t, 3H), 1.74 (m, 1H), 1.81 (d, 2H), 2.18 (m, 1H),2.34 (m, 2H), 2.45 (m, 4H), 3.00 (m, 2H), 3.02 (s, 3H), 3.17 (m, 2H),3.82 (s, 2H), 7.35 (m, 2H), 7.42 (m, 4H), 7.49 (m, 4H).

Example 11

(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8-(5-hexen-1-yl)-8-methyl-8-azoniabicyclo[3.2.1]octanebromide

The title compound was prepared in 29% yield by following the generalexperimental procedure (reaction time=70 hours): LCMS (ES) m/z413 (M)⁺;¹H-NMR(CDCl₃) δ 1.48 (m, 2H), 1.79 (m, 4H), 2.18 (m, 3H), 2.42 (m, 6H),3.00 (m, 5H), 3.22 (m, 2H), 3.82 (s, 2H), 5.05 (m, 2H), 5.86 (m, 1H),7.35 (m, 2H), 7.42 (m, 4H), 7.49 (m, 4H).

Example 12

(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-(4,4,4-trifluorobutyl)-8-azoniabicyclo[3.2.1]octanebromide

The title compound was prepared in 23% yield by following the generalexperimental procedure (reaction time=70 hours): LCMS (ES) m/z 441 (M)⁺;¹H-NMR(CDCl₃) δ 1.80 (d, 2H), 2.03 (m, 2H), 2.19 (m, 1H), 2.41 (m, 8H),3.01 (m, 2H), 3.06 (s, 3H), 3.30 (m, 2H), 3.87 (s, 2H), 7.35 (m, 2H),7.42 (m, 4H), 7.49 (m, 4H).

Example 13

(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-(3-phenylpropyl)-8-azoniabicyclo[3.2.1]octanebromide

The title compound was prepared in 43% yield by following the generalexperimental procedure (reaction time=7 days): LCMS (ES) m/z 449 (M)⁺;¹H-NMR(CDCl₃) δ 1.78 (d, 2H), 2.07 (m, 2H), 2.26 (m, 4H), 2.46 (m, 3H),2.71 (t, 2H), 2.97 (d, 2H), 2.99 (s, 3H), 3.21 (m, 2H), 3.80 (s, 2H),7.25 (m, 2H), 7.31 (m, 4H), 7.41 (m, 5H), 7.47 (m, 4H).

Example 14

(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8-(2-cyclohexylethyl)-8-methyl-8-azoniabicyclo[3.2.1]octanebromide

The title compound was prepared in 21% yield by following the generalexperimental procedure (reaction time=7 days): LCMS (ES) m/z 441 (M)⁺;¹H-NMR(CDCl₃) δ 1.04 (m, 2H), 1.29 (m, 4H), 1.62 (m, 2H), 1.75 (m, 7H),2.18 (m, 1H), 2.34 (m, 4H), 2.49 (m, 2H), 3.00 (m, 5H), 3.24 (m, 2H),3.82 (s, 2H), 7.35 (m, 2H), 7.42 (m, 4H), 7.49 (m, 4H).

Example 15

(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8-(3-cyanopropyl)-8-methyl-8-azoniabicyclo[3.2.1]octanebromide

The title compound was prepared in 48% yield by following the generalexperimental procedure (reaction time=7 days): LCMS (ES) m/z 398 (M)⁺;¹H-NMR(CDCl₃) δ 1.83 (d, 2H), 2.17 (m, 3H), 2.42 (m, 6H), 2.60 (t, 2H),3.01 (m, 2H), 3.06 (s, 3H), 3.32 (m, 2H), 3.87 (s, 2H), 7.35 (m, 2H),7.42 (m, 4H), 7.49 (m, 4H).

Example 16

(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-[2-(methyloxy)ethyl]-8-azoniabicyclo[3.2.1]octanebromide

The title compound was prepared in 24% yield by following the generalexperimental procedure (reaction time=7 days): LCMS (ES) m/z 389 (M)⁺;¹H-NMR(CDCl₃) δ 1.80 (d, 2H), 2.18 (m, 1H), 2.34 (m, 2H), 2.48 (m, 4H),3.00 (m, 2H), 3.10 (s, 3H), 3.38 (s, 3H), 3.50 (m, 2H), 3.81 (m, 2H),3.93 (s, 2H), 7.35 (m, 2H), 7.42 (m, 4H), 7.49 (m, 4H).

Example 17

(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-(2-{[2-(methyloxy)ethyl]oxy}ethyl)-8-azoniabicyclo[3.2.1]octanebromide

The title compound was prepared in 28% yield by following the generalexperimental procedure (reaction time=7 days): LCMS (ES) m/z 433 (M)⁺;¹H-NMR(CDCl₃) δ 1.80 (d, 2H), 2.18 (m, 1H), 2.34 (m, 2H), 2.48 (m, 4H),3.01 (m, 2H), 3.11 (s, 3H), 3.36 (s, 3H), 3.50 (m, 2H), 3.56 (m, 2H),3.64 (m, 2H), 3.91 (m, 2H), 3.96 (s, 2H), 7.35 (m, 2H), 7.43 (m, 4H),7.49 (m, 4H).

Intermediate 2: (3-Endo)-tert-butyl3-(hydroxymethyl)-8-azabicyclo[3.2.1]octane-8-carboxylate

A solution of (3-endo)-(8-benzyl-8-azabicyclo[3.2.1]oct-3-yl)methanol(2.31 g, 10 mmol) in ethanol (45 ml) and 6N HCl (2 ml) containingpalladium hydroxide on carbon (Pearlman's catalyst, 2.6 g, 20% (w/w))was hydrogenated (55 psi H₂) at room temperature for 18 h. Afterfiltration of the catalyst, the filtrate was concentrated under vacuum.The residue was redissolved in EtOH (45 ml) and 6N HCl (2 ml), to whichpalladium hydroxide on carbon (2.6 g) was added. The reaction mixturewas hydrogenated (55 psi H₂) at room temperature for 2 days. Thecatalyst was filtered off over Celite and the filtrate was evaporated.The residue and di-tert-butyl dicarbonate (3.2 g, 15 mmol) weredissolved in 60 ml of dioxane: 1 N NaOH (2:1) and stirred overnight atroom temperature. The solvent was evaporated and the residue partitionedbetween ethyl acetate (3×50 ml) and water (50 ml). The combined organiclayers were dried over Na₂SO₄ and evaporated. The residual oil waspurified by loading onto an aminopropyl SPE cartridge (30 g) and elutingwith DCM (4×30 ml), EA (4×30 ml) and MeOH (2×30 ml). The DCM fractionswere combined and evaporated to give a colourless oil (1.95 g, 81%).LC/MS: 1.65 min (100%), MH+: 242. NMR (CDCl₃): 4.15 ppm (broad, 2H),3.64 ppm (d, 2H), 2.20 ppm (broad, 2H), 1.97 ppm (broad, 2H), 1.85 ppm(m, 1H), 1.60 ppm (m, 2H), 1.40-1.50 ppm (s+broad, 11H)

Intermediate 3: (3-Endo)-tert-butyl3-({[(4-methylphenyl)sulfonyl]oxy}methyl)-8-azabicyclo[3.2.1]octane-8-carboxylate

To a solution of (3-endo)-tert-butyl 3-(hydroxymethyl)-8-azabicyclo[3.2.1] octane-8-carboxylate (840 mg, 3.48 mmol) in CH₂Cl₂ (5 ml) wasadded tosyl chloride (1.33 g, 6.97 mmol) and triethylamine (91 ml, 7.35mmol). The reaction mixture was stirred at room temperature for 16hours. The mixture was diluted with 10 ml of DCM and 2 ml of saturatedNaHCO₃. The organic layer was isolated with a hydrophobic frit andconcentrated under vacuum. The residue was purified by CombiFlash (110 gof Silica, eluting with EA/hexane from 20% to 60% in 65 min.) to affordthe title compound (1.24 g, 90%). LC/MS: m/z, 396 (M+H)+, 2.43 min.

Intermediate 4:(3-Endo)-3-(iodomethyl)-8-(phenylmethyl)-8-azabicyclo[3.2.1]octane

To a solution of iodine (5.08 g, 20 mmol) in DCM (100 ml) was added[8-(phenylmethyl)-8-azabicyclo[3.2.1]oct-3-yl]methanol (2.31 g, 10 mmol)and a polymer supported PPh₃ (9.0 g, 2.21 mmol/g, 20 mmol). Afterstirring for 17 hours, the reaction mixture was filtered andconcentrated under vacuum to afford the title compound (3.07 g, 90%);LC/MS: m/z, 342 (M+H)+, 1.42 min.

Intermediate 5:(3-Endo)-2,2-diphenyl-3-[8-(phenylmethyl)-8-azabicyclo[3.2.1]oct-3-yl]propanenitrile

A mixture of(3-endo)-3-(iodomethyl)-8-(phenylmethyl)-8-azabicyclo[3.2.1]octane (2.1g, 6.16 mmol), Ph₂CHCN (3.56 g, 18.5 mmol) and NaH (0.44 g, 18.5 mmol)in a 100 ml round bottom flask was flushed with Argon for 30 min beforeslowly adding DMF (20 ml) under agitation. The resulting mixture wasstirred at room temperature for 60 minutes then diluted with EA (200 ml)and water (100 ml). The aqueous layer was separated and extracted withEA (2×100 ml). The combined organic layers were evaporated, and theresidue was purified by CombiFlash (110 g of Silica, eluting withEA/hexane from 20% to 60% in 65 min.) to afford the title compound (1.50g, 60%); LC/MS: m/z, 407 (M+H)+, 1.90 min; 1H-NMR(CDCl3) 1.29 (d, 2H),1.74 (m, 2H), 2.04-2.15 (m, 5H), 2.66 (s, 2H), 3.15 (s, 2H), 3.52 (s,2H), 7.24-7.41 (m, 15H).

Intermediate 6:(3-Endo)-3-(8-azabicyclo[3.2.1]oct-3-yl)-2,2-diphenylpropanenitrile

a) From(3-Endo)-2,2-diphenyl-3-[8-(phenylmethyl)-8-azabicyclo[3.2.1]oct-3-yl]propanenitrile

To a solution of(3-endo)-2,2-diphenyl-3-[8-(phenylmethyl)-8-azabicyclo[3.2.1]oct-3-yl]propanenitrile(82 mg, 0.2 mmol) in 1.5 ml of DCE at 0° C., was slowly added1-chloroethyl chloroformate (115 mg, 0.8 mmol). The mixture was heatedin a microwave at 140° C. for 80 min. LC/MS showed ˜70% of the desiredproduct and 13% of the starting material. Further extension of thereaction time didn't improve the yield as assessed by LC/MS. Thereaction mixture was quenched with 1 ml of MeOH and allowed to heat atreflux for 1 h. After evaporation of the solvent, the residue waspurified by reverse-phase Gilson HPLC, eluting with eluting withacetonitrile/water/0.1% TFA (10/90 to 70/30, v/v, over 12 min), to givethe title compound (56 mg, 88%). LC/MS: m/z, 317 (M+H), 1.54 min.

b) From (3-endo)-tert-butyl3-({[(4-methylphenyl)sulfonyl]oxy}methyl)-8-azabicyclo[3.2.1]octane-8-carboxylate

To a mixture of (3-endo)-tert-butyl3-({[(4-methylphenyl)sulfonyl]oxy}methyl)-8-azabicyclo[3.2.1]octane-8-carboxylate(0.46 g, 1.16 mmol), Ph₂CHCN (0.67 g, 3.31 mmol) and NaH (84 mg, 3.5mmol) was slowly added 6 ml of dry DMF under Argon for 10 min. Theresulting mixture was heated in a microwave reactor at 220° C. for 60minutes. The solution was filtered and purified by Gilson reverse-phaseHPLC eluting with acetonitrile/water/0.1% TFA (10/90 to 70/30, v/v, over12 min), to give the title compound as a TFA salt (400 mg, 80%). LC/MS:m/z, 317 (M+H), 1.64 min.

Intermediate 7:(3-Endo)-3-[8-(5-hexen-1-yl)-8-azabicyclo[3.2.1]oct-3-yl]-2,2-diphenylpropanenitrile

To a solution of(3-endo)-3-(8-azabicyclo[3.2.1]oct-3-yl)-2,2-diphenylpropanenitrile (TFAsalt, 256 mg, 0.59 mmol) in 10 ml of dry acetonitrile, was added6-bromo-1-hexene (108 mg, 0.65 mmol) and K₂CO₃ (447 mg, 3.24 mmol). Themixture was heated at reflux for 16 hours, filtered and concentratedunder vacuum. The crude residue was purified by loading onto a 10 gaminopropyl SPE cartridge, eluting sequentially with toluene (3×5 ml)and EA (3×5 ml). The toluene fractions 2 and 3 and the EA fractions 1and 2 were combined and evaporated to give the title compound as paleyellow oil (150 mg, 64%). LC/MS: m/z, 399 (M+H), 1.93 min.

Example 18N-(Endo)-(3-endo)-3-(2-cyano-2,2-diphenylethyl)-(8-endo)-8-(5-hexen-1-yl)-8-methyl-8-azoniabicyclo[3.2.1]octanebromide

To a solution of(3-endo)-3-[8-(5-hexen-1-yl)-8-azabicyclo[3.2.1]oct-3-yl]-2,2-diphenylpropanenitrile(300 mg, 0.75 mmol) in 1 ml of acetone, was added bromomethane int-butyl ether (2 M, 7.5 ml, 15 mmol) and K₂CO₃ (104 mg, 0.75 mmol)followed by stirring at room temperature for 16 hours. After addition ofDCM (20 ml) and filtration, the filtrate was concentrated under vacuumto give a crude product, which was recrystallized with DCM/EA/hexane togive ˜250 mg of a white solid, containing ˜5% exo isomer as assessed byNMR. Another recrystallization under the same conditions gave the pureendo isomer (148 mg, 40%). LC/MS: m/z, 413 (M+H), 1.93 min.

Intermediate 8:(3-Endo)-2,2-diphenyl-3-(8-{2-[(phenylmethyl)oxy]ethyl}-8-azabicyclo[3.2.1]oct-3-yl)propanenitrile

Following the standard procedure outlined for Intermediate 7,(3-endo)-3-(8-azabicyclo[3.2.1]oct-3-yl)-2,2-diphenylpropanenitrile (TFAsalt, 160 mg, 0.37 mmol) was reacted with 2-bromoethyl phenylmethylether (80 mg, 0.37 mmol) to give the title compound (150 mg, 90%).LC/MS: m/z, 451 (M+H), 1.93 min.

Intermediate 9(3-Endo)-2,2-diphenyl-3-[8-(3-phenylpropyl)-8-azabicyclo[3.2.1]oct-3-yl]propanenitrile

Following the standard procedure outlined for Intermediate 7,(3-endo)-3-(8-azabicyclo[3.2.1]oct-3-yl)-2,2-diphenylpropanenitrile (TFAsalt, 160 mg, 0.37 mmol) was reacted with 3-bromopropylbezene (74 mg,0.37 mmol) to give the title compound (140 mg, 87%). LC/MS: m/z, 435(M+H), 1.85 min.

Intermediate 10(3-Endo)-2,2-diphenyl-3-{8-[3-(phenyloxy)propyl]-8-azabicyclo[3.2.1]oct-3-yl}propanenitrile

Following the standard procedure outlined for Intermediate 7,(3-endo)-3-(8-azabicyclo[3.2.1]oct-3-yl)-2,2-diphenylpropanenitrile (TFAsalt, 160 mg, 0.37 mmol) was reacted with 3-bromopropyl phenyl ether (80mg, 0.37 mmol) to give the title compound (150 mg, 90%). LC/MS: m/z, 451(M+H), 2.00 min.

Example 19N-(Endo)-(3-endo)-(2-cyano-2,2-diphenylethyl)-(8-endo)-methyl-8-{2-[(phenylmethyl)oxy]ethyl}-8-azoniabicyclo[3.2.1]octanebromide

Following the standard procedure outlined in Example 18,(3-endo)-2,2-diphenyl-3-(8-{2-[(phenylmethyl)oxy]ethyl}-8-azabicyclo[3.2.1]oct-3-yl)propanenitrile(150 mg, 0.33 mmol) was reacted with bromomethane (2M, 3.3 ml, 6.6 mmol)to give the title compound (115 mg, 63%). LC/MS: m/z, 465 (M+H), 2.01min.

Example 20N-(Endo)-(3-endo)-(2-cyano-2,2-diphenylethyl)-8-methyl-8-(3-phenylpropyl)-8-azoniabicyclo[3.2.1]octanebromide

Following the standard procedure outlined in Example 18,(3-Endo)-2,2-diphenyl-3-[8-(3-phenylpropyl)-8-azabicyclo[3.2.1]oct-3-yl]propanenitrile(140 mg, 0.3 mmol) was reacted with bromomethane (2M, 3.2 ml, 6.4 mmol)to give the title compound (100 mg, 59%). LC/MS: m/z, 449 (M+H), 2.02min.

Example 21N-(Endo)-(3-endo)-(2-cyano-2,2-diphenylethyl)-8-methyl-8-[3-(phenyloxy)propyl]-8-azoniabicyclo[3.2.1]octanebromide

Following the standard procedure outlined Example 18,(3-endo)-3-(8-azabicyclo[3.2.1]oct-3-yl)-2,2-diphenylpropanenitrile (150mg, 0.33 mmol) was reacted with bromomethane (2M, 3.3 ml, 6.6 mmol) togive the title compound (115 mg, 63%). LC/MS: m/z, 465 (M+H), 2.04 min.

Abbreviations

-   BOC tert-butyloxycarbonyl-   DCM Dichloromethane-   DMF Dimethylformamide-   DMSO Dimethylsulfoxide-   ES Electrospray ionization-   HPLC High pressure liquid chromatography-   LCMS Liquid chromatography mass spectrometry-   NMR Nuclear magnetic resonance-   SPE Solid phase extraction-   TEA Triethylamine-   TFA Trifluoroacetic acid-   THF Tetrahydrofuran-   TLC Thin layer chromatography

BIOLOGICAL EXAMPLES

The inhibitory effects of compounds at the M₃ mAChR of the presentinvention are determined by the following in vitro and in vivo assay:

Analysis of Inhibition of Receptor Activation by Calcium Mobilization:

Stimulation of mAChRs expressed on CHO cells were analyzed by monitoringreceptor-activated calcium mobilization as previously described¹⁰. CHOcells stably expressing M₃ mAChRs were plated in 96 well blackwall/clear bottom plates. After 18 to 24 hours, media was aspirated andreplaced with 100 μl of load media (EMEM with Earl's salts, 0.1%RIA-grade BSA (Sigma, St. Louis Mo.), and 4 μM Fluo-3-acetoxymethylester fluorescent indicator dye (Fluo-3 AM, Molecular Probes, Eugene,Oreg.) and incubated 1 hr at 37° C. The dye-containing media was thenaspirated, replaced with fresh media (without Fluo-3 AM), and cells wereincubated for 10 minutes at 37° C. Cells were then washed 3 times andincubated for 10 minutes at 37° C. in 100 μl of assay buffer (0.1%gelatin (Sigma), 120 mM NaCl, 4.6 mM KCl, 1 mM KH₂ PO₄, 25 mM NaH CO₃,1.0 mM CaCl₂, 1.1 mM MgCl₂, 11 mM glucose, 20 mM HEPES (pH 7.4)). 50 μlof compound (1×10⁻¹¹-1×10⁻⁵ M final in the assay) was added and theplates were incubated for 10 min. at 37° C. Plates were then placed intoa fluorescent light intensity plate reader (FLIPR, Molecular Probes)where the dye loaded cells were exposed to excitation light (488 nm)from a 6 watt argon laser. Cells were activated by adding 50 μl ofacetylcholine (0.1-10 nM final), prepared in buffer containing 0.1% BSA,at a rate of 50 μl/sec. Calcium mobilization, monitored as change incytosolic calcium concentration, was measured as change in 566 nmemission intensity. The change in emission intensity is directly relatedto cytosolic calcium levels¹¹. The emitted fluorescence from all 96wells is measured simultaneously using a cooled CCD camera. Data pointsare collected every second. This data was then plotting and analyzedusing GraphPad PRISM software.

Methacholine-Induced Bronchoconstriction

Airway responsiveness to methacholine was determined in awake,unrestrained BalbC mice (n=6 each group). Barometric plethysmography wasused to measure enhanced pause (Penh), a unitless measure that has beenshown to correlate with the changes in airway resistance that occurduring bronchial challenge with methacholine¹². Mice were pretreatedwith 50 μl of compound (0.003-10 μg/mouse) in 50 μl of vehicle (10%DMSO) intranasally, and were then placed in the plethysmography chamber.Once in the chamber, the mice were allowed to equilibrate for 10 minbefore taking a baseline Penh measurement for 5 minutes. Mice were thenchallenged with an aerosol of methacholine (10 mg/ml) for 2 minutes.Penh was recorded continuously for 7 min starting at the inception ofthe methacholine aerosol, and continuing for 5 minutes afterward. Datafor each mouse were analyzed and plotted by using GraphPad PRISMsoftware.

Muscarinic Receptor Radioligand Binding Assays

Radioligand binding studies using 0.5 nM [³H]-N-methyl scopolamine (NMS)in a SPA format is used to assess binding of muscarinic antagonists toM₁, M₂, M₃, M₄ and M₅ muscarinic acetylcholine receptors. In a 96-wellplate, the SPA beads are pre-incubated with receptor-containing membranefor 30 min at 4° C. Then 50 mM HEPES and the test compound are added andincubated at room temperature (shaking) for 2 hours. The beads are thenspun down and counted using a scintillation counter.

Evaluation of Potency and Duration of Action in Isolated Guinea PigTrachea

Tracheae were removed from adult male Hartely guinea pigs (CharlesRiver, Raleigh, N.C.; 400-600 grams) and placed into modifiedKrebs-Henseleit solution. Composition of the solution was (mM): NaCl113.0, KCl 4.8, CaCl₂ 2.5, KH₂PO₄ 1.2, MgSO₄ 1.2, NaHCO₃ 25.0 anddextrose 11.0. which was gassed with 95% O₂: 5% CO₂ and maintained at37° C. Each trachea was cleaned of adherent tissue and openedlengthwise. Epithelium was removed by gently rubbing the luminal surfacewith a cotton-tipped applicator. Individual strips were cut,approximately 2 cartilage rings in width, and suspended via silk suturein 10-ml water-jacketed organ baths containing Krebs-Henseleit solutionand connected to Grass FT03C force-displacement transducers. Mechanicalresponses were recorded isometrically by MP100WS/Acknowledge dataacquisition system (BIOPAC Systems, Goleta, Calif., www.biopac.com) runon Apple G4 computers. The tissues were equilibrated under a restingtension of 1.5 g, determined to be optimal by length-tension evaluation,and washed with Krebs-Henseleit solution every 15 minutes for one hour.After the equilibration period pulmonary tissues were contracted with 10uM carbachol until reaching plateau, which served as a referencecontraction for data analysis. Tissues were then rinsed every 15 minutesover 1 hour until reaching baseline tone. The preparations were thenleft for at least 30 minutes before the start of the experiment.

Concentration-response curves were obtained by a cumulative addition ofcarbachol in half-log increments (Van Rossum, 1963, Arch. Int.Pharmacodyn., 143:299), initiated at 1 nM. Each concentration was leftin contact with the preparation until the response plateaued before theaddition of the subsequent carbachol concentration. Paired tissues wereexposed to mAChR antagonist compounds or vehicle for 30 min beforecarbachol cumulative concentration-response curves were generated. Alldata is given as mean±standard error of the mean (s.e.m.) with n beingthe number of different animals.

For superfusion (duration of action) studies, the tissues werecontinuously superfused with Krebs-Henseleit solution at 2 ml/min forthe duration of the experiment. Stock solutions of agonist andantagonist were infused (0.02 ml/min) via 22-gauge needle inserted intothe superfusion tubing. Mechanical responses were recorded isometricallyusing a commercially-available data acquisition system(MP100WS/Acknowledge; BIOPAC Systems, Goleta, Calif., www.biopac.com)interfaced with a Macintosh G4 computer (Apple, Cupertino, Calif.www.apple.com). The tissues were suspended under an optimal restingtension of 1.5 g. After a 60 min equilibration period, the tissues werecontracted with carbachol (1 uM) for the duration of the experiment.Upon reaching a sustained contraction isoproterenol (10 uM) wasadministered to maximally relax the tissue, and this change served as areference. Isoproterenol exposure was halted and the carbachol-inducedtension allowed to recover. Muscarinic receptor antagonists infused at asingle concentration per tissue until a sustained level of inhibitionwas attained. The compound was then removed and, once again, thecarbachol-induced tension was allowed to recover.

The following parameters were determined for each concentration ofantagonist, and expressed as the mean±S.E.M. for n individual animals.Inhibition of the carbachol-induced contraction was expressed as apercent of the reference response (isoproterenol) and the time requiredto reach one-half of this relaxation was measured (onset of response).The tension recovery following removal of the compound was determined aswas the time required to reach one-half of the maximum tension recovery(offset of response). At 60 and 180 minutes after removal of theantagonist the remaining level of inhibition was determined andexpressed as a percent of the isoproterenol reference.

Antagonist concentration-response curves were obtained by plotting themaximal relaxation data at 0, 60 and 180-min following antagonistwithdrawal. Recovery, termed shift, was calculated from the ratio of the0-min inhibition curve IC₅₀ and the concentration of compound yielding asimilar tension recovery at 60 and 180 minutes.

Halftimes for onset and offset of response were plotted vs.corresponding concentration and the data were fit with non-linearregression. These values were extrapolated at the IC₅₀ (determined fromthe inhibition concentration-response curve) and designated Ot₅₀ (timerequired, at the IC₅₀ concentration, to reach half of the onsetresponse) and Rt50 (time required, at the IC₅₀ concentration, to reachhalf of the recovery response).

Formulation-Administration

Accordingly, the present invention further provides a pharmaceuticalformulation comprising a compound of formula (I), or a pharmaceuticallyacceptable salt, solvate, or physiologically functional derivative(e.g., salts and esters) thereof, and a pharmaceutically acceptablecarrier or excipient, and optionally one or more other therapeuticingredients.

Hereinafter, the term “active ingredient” means a compound of formula(I), or a pharmaceutically acceptable salt, solvate, or physiologicallyfunctional derivative thereof.

Compounds of formula (I) will be administered via inhalation via themouth or nose.

Dry powder compositions for topical delivery to the lung by inhalationmay, for example, be presented in capsules and cartridges of for examplegelatine, or blisters of for example laminated aluminium foil, for usein an inhaler or insufflator. Powder blend formulations generallycontain a powder mix for inhalation of the compound of the invention anda suitable powder base (carrier/diluent/excipient substance) such asmono-, di- or poly-saccharides (e.g., lactose or starch), organic orinorganic salts (e.g., calcium chloride, calcium phosphate or sodiumchloride), polyalcohols (e.g., mannitol), or mixtures thereof,alternatively with one or more additional materials, such additivesincluded in the blend formulation to improve chemical and/or physicalstability or performance of the formulation, as discussed below, ormixtures thereof. Use of lactose is preferred. Each capsule or cartridgemay generally contain between 20 μg-10 mg of the compound of formula (I)optionally in combination with another therapeutically activeingredient. Alternatively, the compound of the invention may bepresented without excipients, or may be formed into particles comprisingthe compound, optionally other therapeutically active materials, andexcipient materials, such as by co-precipitation or coating.

Suitably, the medicament dispenser is of a type selected from the groupconsisting of a reservoir dry powder inhaler (RDPI), a multi-dose drypowder inhaler (MDPI), and a metered dose inhaler (MDI).

By reservoir dry powder inhaler (RDPI) it is meant as an inhaler havinga reservoir form pack suitable for comprising multiple (un-metereddoses) of medicament in dry powder form and including means for meteringmedicament dose from the reservoir to a delivery position. The meteringmeans may for example comprise a metering cup or perforated plate ,which is movable from a first position where the cup may be filled withmedicament from the reservoir to a second position where the meteredmedicament dose is made available to the patient for inhalation.

By multi-dose dry powder inhaler (MDPI) is meant an inhaler suitable fordispensing medicament in dry powder form, wherein the medicament iscomprised within a multi-dose pack containing (or otherwise carrying)multiple, define doses (or parts thereof) of medicament. In a preferredaspect, the carrier has a blister pack form, but it could also, forexample, comprise a capsule-based pack form or a carrier onto whichmedicament has been applied by any suitable process including printing,painting and vacuum occlusion.

The formulation can be pre-metered (e.g. as in Diskus, see GB 2242134 orDiskhaler, see GB 2178965, 2129691 and 2169265) or metered in use (e.g.as in Turbuhaler, see EP 69715). An example of a unit-dose device isRotahaler (see GB 2064336). The Diskus inhalation device comprises anelongate strip formed from a base sheet having a plurality of recessesspaced along its length and a lid sheet hermetically but peelably sealedthereto to define a plurality of containers, each container havingtherein an inhalable formulation containing a compound of formula (I)preferably combined with lactose. Preferably, the strip is sufficientlyflexible to be wound into a roll. The lid sheet and base sheet willpreferably have leading end portions which are not sealed to one anotherand at least one of the said leading end portions is constructed to beattached to a winding means. Also, preferably the hermetic seal betweenthe base and lid sheets extends over their whole width. The lid sheetmay preferably be peeled from the base sheet in a longitudinal directionfrom a first end of the said base sheet.

In one aspect, the multi-dose pack is a blister pack comprising multipleblisters for containment of medicament in dry powder form. The blistersare typically arranged in regular fashion for ease of release ofmedicament therefrom.

In one aspect, the multi-dose blister pack comprises plural blistersarranged in generally circular fashion on a disk-form blister pack. Inanother aspect, the multi-dose blister pack is elongate in form, forexample comprising a strip or a tape.

Preferably, the multi-dose blister pack is defined between two memberspeelably secured to one another. U.S. Pat. Nos. 5,860,419, 5,873,360 and5,590,645 describe medicament packs of this general type. In thisaspect, the device is usually provided with an opening stationcomprising peeling means for peeling the members apart to access eachmedicament dose. Suitably, the device is adapted for use where thepeelable members are elongate sheets which define a plurality ofmedicament containers spaced along the length thereof, the device beingprovided with indexing means for indexing each container in turn. Morepreferably, the device is adapted for use where one of the sheets is abase sheet having a plurality of pockets therein, and the other of thesheets is a lid sheet, each pocket and the adjacent part of the lidsheet defining a respective one of the containers, the device comprisingdriving means for pulling the lid sheet and base sheet apart at theopening station.

By metered dose inhaler (MDI) it is meant a medicament dispensersuitable for dispensing medicament in aerosol form, wherein themedicament is comprised in an aerosol container suitable for containinga propellant-based aerosol medicament formulation. The aerosol containeris typically provided with a metering valve, for example a slide valve,for release of the aerosol form medicament formulation to the patient.The aerosol container is generally designed to deliver a predetermineddose of medicament upon each actuation by means of the valve, which canbe opened either by depressing the valve while the container is heldstationary or by depressing the container while the valve is heldstationary.

Spray compositions for topical delivery to the lung by inhalation mayfor example be formulated as aqueous solutions or suspensions or asaerosols delivered from pressurised packs, such as a metered doseinhaler, with the use of a suitable liquefied propellant. Aerosolcompositions suitable for inhalation can be either a suspension or asolution and generally contain the compound of formula (I) optionally incombination with another therapeutically active ingredient and asuitable propellant such as a fluorocarbon or hydrogen-containingchlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes,e.g. dichlorodifluoromethane, trichlorofluoromethane,dichlorotetra-fluoroethane, especially 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3-heptafluoro-n-propane or a mixture thereof. Carbon dioxideor other suitable gas may also be used as propellant. The aerosolcomposition may be excipient free or may optionally contain additionalformulation excipients well known in the art such as surfactants e.g.oleic acid or lecithin and co solvents e.g. ethanol. Pressurizedformulations will generally be retained in a canister (e.g. an aluminiumcanister) closed with a valve (e.g. a metering valve) and fitted into anactuator provided with a mouthpiece.

Medicaments for administration by inhalation desirably have a controlledparticle size. The optimum aerodynamic particle size for inhalation intothe bronchial system for localized delivery to the lung is usually 1-10μm, preferably 2-5 μm. The optimum aerodynamic particle size forinhalation into the alveolar region for achieving systemic delivery tothe lung is approximately 0.5-3 μm, preferably 1-3 μm. Particles havingan aerodynamic size above 20 μm are generally too large when inhaled toreach the small airways. Average aerodynamic particle size of aformulation may measure by, for example cascade impaction. Averagegeometric particle size may be measured, for example by laserdiffraction, optical means.

To achieve a desired particle size, the particles of the activeingredient as produced may be size reduced by conventional means eg bycontrolled crystallization, micronisation or nanomilling. The desiredfraction may be separated out by air classification. Alternatively,particles of the desired size may be directly produced, for example byspray drying, controlling the spray drying parameters to generateparticles of the desired size range. Preferably, the particles will becrystalline, although amorphous material may also be employed wheredesirable. When an excipient such as lactose is employed, generally, theparticle size of the excipient will be much greater than the inhaledmedicament within the present invention, such that the “coarse” carrieris non-respirable. When the excipient is lactose it will typically bepresent as milled lactose, wherein not more than 85% of lactoseparticles will have a MMD of 60-90 μm and not less than 15% will have aMMD of less than 15 μm. Additive materials in a dry powder blend inaddition to the carrier may be either respirable, i.e., aerodynamicallyless than 10 microns, or non-respirable, i.e., aerodynamically greaterthan 10 microns.

Suitable additive materials which may be employed include amino acids,such as leucine; water soluble or water insoluble, natural or syntheticsurfactants, such as lecithin (e.g., soya lecithin) and solid statefatty acids (e.g., lauric, palmitic, and stearic acids) and derivativesthereof (such as salts and esters); phosphatidylcholines; sugar esters.Additive materials may also include colorants, taste masking agents(e.g., saccharine), anti-static-agents, lubricants (see, for example,Published PCT Patent Appl. No. WO 87/905213, the teachings of which areincorporated by reference herein), chemical stabilizers, buffers,preservatives, absorption enhancers, and other materials known to thoseof ordinary skill.

Sustained release coating materials (e.g., stearic acid or polymers,e.g. polyvinyl pyrolidone, polylactic acid) may also be employed onactive material or active material containing particles (see, forexample, U.S. Pat. No. 3,634,582, GB 1,230,087, GB 1,381,872, theteachings of which are incorporated by reference herein).

Intranasal sprays may be formulated with aqueous or non-aqueous vehicleswith the addition of agents such as thickening agents, buffer salts oracid or alkali to adjust the pH, isotonicity adjusting agents oranti-oxidants.

Solutions for inhalation by nebulation may be formulated with an aqueousvehicle with the addition of agents such as acid or alkali, buffersalts, isotonicity adjusting agents or antimicrobials. They may besterilised by filtration or heating in an autoclave, or presented as anon-sterile product.

Preferred unit dosage formulations are those containing an effectivedose, as herein before recited, or an appropriate fraction thereof, ofthe active ingredient.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer or step or group of integers but not to theexclusion of any other integer or step or group of integers or steps.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

The above description fully discloses the invention including preferredembodiments thereof. Modifications and improvements of the embodimentsspecifically disclosed herein are within the scope of the followingclaims. Without further elaboration, it is believed that one skilled inthe art can, using the preceding description, utilize the presentinvention to its fullest extent. Therefore the Examples herein are to beconstrued as merely illustrative and not a limitation of the scope ofthe present invention in any way. The embodiments of the invention inwhich an exclusive property or privilege is claimed are defined asfollows.

1. A method of inhibiting the binding of acetylcholine to aacetylcholine receptor in the respiratory tract of a mammal in needthereof, which comprises contacting the acetylcholine receptor with aneffective amount of a compound of formula (I)

wherein: R1⁻ represents an anion associated with the positive charge ofthe N atom; R2 is aryl; R3 is (C₁-C₆)alkyl-O-(C₁-C₆)aryl, via inhalationby the mouth or nose of the mammal.
 2. The method according to claim 1wherein the H atom indicated is in the exo position.
 3. The methodaccording to claim 1 wherein R1⁻ is selected from the group consistingof chloride, bromide, iodide, sulfate, benzene sulfonate and toluenesulfonate.
 4. The method according to claim 1 wherein R2 is phenyl. 5.The method according to claim 1 wherein R3 is(C₃alkyl)—O—(C₂alkyl)-aryl.
 6. The method according to claim 5 whereinthe aryl is phenyl.
 7. The method according to claim 1 wherein thecompound of formula (I) isN-(Endo)-(3-endo)-(2-cyano-2,2-diphenylethyl)-(8-endo)-methyl-8-{2-[(phenylmethyl)oxy]ethyl}-8-azoniabicyclo[3.2.1]octaneand a pharmaceutically acceptable anion thereof.
 8. The method accordingclaim 1 wherein the compound of formula (I) isN-(Endo)-(3-endo)-(2-cyano-2,2-diphenylethyl)-(8-endo)-methyl-8-{2-[(phenylmethyl)oxy]ethyl}-8-azoniabicyclo[3.2.1]octanebromide.
 9. A method of inhibiting the binding of acetylcholine to an M₃muscarinic acetylcholine receptor in the respiratory tract of a mammalin need thereof, which comprises contacting the M₃ muscarinicacetylcholine receptor with an effective amount of a compound of formula(I)

wherein: R1⁻ represents an anion associated with the positive charge ofthe N atom; R2 is aryl; R3 is (C₁-C₆)alkyl-O-(C₁-C₆)aryl, via inhalationby the mouth or nose of the mammal:
 14. The method according to claim 13wherein the binding of the M3 muscarinic acetylcholine receptor isuseful in the treatment of chronic obstructive lung disease, chronicbronchitis, asthma, chronic respiratory obstruction, pulmonary fibrosis,pulmonary emphysema or allergic rhinitis.
 15. A method according toclaim 14 wherein the method of contacting is via the nose.
 16. A methodaccording to claim 14 wherein the method of contacting is via the mouth.17. A method according to claim 14 wherein the method of contacting isby administration of the compound from a medicament dispenser selectedfrom a reservoir dry powder inhaler, a multi-dose dry powder inhaler ora metered dose inhaler.
 18. A method according to claim 14 wherein thecompound is administered to a human and has a duration of action of 12hours or more and the mammal is a human.
 19. The method according toclaim 13 wherein the compound of formula (I) isN-(Endo)-(3-endo)-(2-cyano-2,2-diphenylethyl)-(8-endo)-methyl-8-{2-[(phenylmethyl)oxy]ethyl}-8-azoniabicyclo[3.2.1]octaneand a pharmaceutically acceptable anion thereof.
 20. The methodaccording claim 13 wherein the compound of formula (I) isN-(Endo)-(3-endo)-(2-cyano-2,2-diphenylethyl)-(8-endo)-methyl-8-{2-[(phenylmethyl)oxy]ethyl}-8-azoniabicyclo[3.2.1]octanebromide.
 21. A method of treating chronic obstructive lung disease,chronic bronchitis, asthma, chronic respiratory obstruction, pulmonaryfibrosis, pulmonary emphysema or allergic rhinitis in a human in needthereof, comprising administering to said human by inhalation via themouth or nose, an effective amount of a compound of formula (I)

wherein: R1⁻ represents an anion associated with the positive charge ofthe N atom; R2 is aryl; R3 is (C₁-C₆)alkyl-O-(C₁-C₆)aryl.
 22. The methodaccording to claim 21 wherein the compound of formula (I) isN-(Endo)-(3-endo)-(2-cyano-2,2-diphenylethyl)-(8-endo)-methyl-8-{2-[(phenylmethyl)oxy]ethyl}-8-azoniabicyclo[3.2.1]octaneand a pharmaceutically acceptable anion thereof.
 23. The methodaccording claim 21 wherein the compound of formula (I) isN-(Endo)-(3-endo)-(2-cyano-2,2-diphenylethyl)-(8-endo)-methyl-8-{2-[(phenylmethyl)oxy]ethyl}-8-azoniabicyclo[3.2.1]octanebromide.